Ganglioside Gm1 is the cell surface receptor for cholera toxin. The oligosaccharide chain of Gm1 is recognized by the B or binding subunits of the toxin whereas the A subunit of the toxin activates adenylate cyclase by ADP-ribosylation of the stimulatory G protein of the cyclase system. Less is known about the role(s) that the lipid moiety of GM1 plays in toxin action. We synthesized derivatives of GM1 in which the oligosaccharide was coupled to different lipids such as phospholipids, cholesterol and aliphatic amines. We tested the derivatives on rat glioma C6 cells which are deficient in GM1, bind only traces of the toxin and are poorly responsive to it. C6 cells incorporated the neoglycolipids into the plasma membranes as measured by the increase in cholera toxin binding. However, neoglycolipids consisting of GM1-oligosaccharide attached to short chain aliphatic amines were not taken up by the cells; at least a dodecylamine was required. The neoglycolipids also conferred enhanced cholera toxin responsiveness on the cells as measured by increased stimulation of adenylate cyclase. There were differences among the neoglycolipids in terms of the amounts of cyclic AMP formed per amount of toxin bound to the treated cells. Those derived from long-chain aliphatic amines and cholesterol were more efficient than GM1 as toxin receptors whereas those derived from phospholipids were less efficient than GM1. When the distance between the oligosaccharide and the phospholipid was increased by adding spacers, the efficiency as receptors decreased. Thus, the lipid moiety of GM1 is important for the action of cholera toxin and these neoglycolipids may be useful as probes of GM1 bioactivity in other systems such as the neuritogenic and neuronotrophic effects of the ganglioside.